Bacterial flagellar motor PL-ring disassembly subcomplexes are widespread and ancient

Abstract

The bacterial flagellum is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic subcomplex comprising the decorated P and L rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here, we show that this subcomplex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this subcomplex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P and L rings is to seal the outer membrane after motor disassembly.

Keywords: cryo-electron tomography; evolution; flagellar motor.

Fig. 1.

A taxonomic tree of representative bacterial species. The species where PL-subcomplexes were previously reported are highlighted in gray (all in the Gammaproteobacteria class), while species with PL-subcomplexes identified in this study are highlighted in yellow. PVC, Planctomycetes–Verrucomicrobia–Chlamydiae.

Fig. 2.

Cryo-ET of the sheathed Gammaproteobacteria Vibrio species. (A–C) Slices through electron cryo-tomograms of V. cholerae (A), V. fischeri (B), and V. harveyi (C), highlighting the presence of a single polar sheathed flagellum in the three species (red arrows). (Scale bars: 100 nm.) (D–F) Slices through electron cryo-tomograms of V. cholerae (D), V. fischeri (E), and V. harveyi (F), highlighting the presence of flagellar disassembly PL-subcomplexes (blue circles). (Scale bars: 100 nm.) (G–I) Central slices through subtomogram averages of PL-subcomplexes in V. cholerae (G), V. fischeri (H), and V. harveyi (I). Purple arrows highlight the presence of intact outer membrane (OM) above the PL-subcomplexes. Yellow arrows indicate the proteinaceous plug inside the P ring. Blue arrows in I highlight the presence of an extracellular ring density in the average of V. harveyi. (Scale bars: 20 nm.) IM, inner membrane.

Fig. 3.

Cryo-ET of the Alphaproteobacteria species. (A–C) Slices through electron cryo-tomograms of A. tumefaciens highlighting the presence of flagellar disassembly PL-subcomplexes with zoom-ins (Insets) of these subcomplexes present in the red squares. (Scale bars: 100 nm.) (D) Central slice through a subtomogram average of PL-subcomplexes in A. tumefaciens. (Scale bar: 20 nm.) (E–G) Same as in A–C, but for H. neptunium. (Scale bars: 100 nm.) (H) Central slice through a subtomogram average of PL-subcomplexes in H. neptunium. (Scale bar: 10 nm.) (I–K) Same as in A–C, but for C. crescentus. (Scale bars: 50 nm.) (L) Central slice through a subtomogram average of PL-subcomplexes in C. crescentus. (Scale bar: 10 nm.) IM, inner membrane; OM, outer membrane.

Fig. 4.

Cryo-ET of Betaproteobacteria, Epsilonproteobacteria, and Firmicutes. (A–C) Slices through electron cryo-tomograms of H. gracilis highlighting the presence of flagellar disassembly PL-subcomplexes with zoom-ins (Insets) of these subcomplexes present in the red squares. (Scale bars: 50 nm.) (D) Central slice through a subtomogram average of PL-subcomplexes in H. gracilis. (Scale bar: 20 nm.) (E) A Slice through electron cryo-tomogram of C. jejuni highlighting the presence of a flagellar disassembly PL-subcomplex (red square). (Scale bar: 50 nm.) (F) A zoom-in of the area enclosed in the red square in E. (Scale bar: 20 nm.) (G and H) Slices through electron cryo-tomograms of A. longum highlighting the presence of flagellar disassembly PL-subcomplexes with zoom-ins (Insets) of these subcomplexes present in the red squares. (Scale bars: 50 nm.)

Fig. 5.

Implicit phylogenetic analysis of bacterial L- and P-ring proteins. (A) A scatter plot of pairwise sequence distance of the 15 investigated species in this study based on concatenated 25 reference proteins and the L-ring protein FlgH. Some examples of pairwise species comparisons are annotated in the plot for the sake of clarity. (B) Same as in A, but with the P-ring protein FlgI. Plots shown in A and B are made with the primary copies of P. shigelloides and S. putrefaciens FlgI and FlgH proteins. For similar plots with the secondary copies of FlgI and FlgH in these two species, see SI Appendix, Figs. S4 and S5. The x and y axes in these plots have arbitrary units.